RESEARCH ARTICLE

COPI-mediated retrieval of SCAP is crucial for regulatinglipogenesis under basal and sterol-deficient conditionsKouhei Takashima1, Akina Saitoh1, Teruki Funabashi1, Shohei Hirose1, Chikako Yagi1, Shohei Nozaki1,Ryuichiro Sato2, Hye-Won Shin1,3 and Kazuhisa Nakayama1,*

ABSTRACTRetrograde trafficking from the Golgi complex to endoplasmicreticulum (ER) through COPI-coated vesicles has been implicatedin lipid homeostasis. Here, we find that a block in COPI-dependentretrograde trafficking promotes processing and nuclear translocationof sterol regulatory element binding proteins (SREBPs), andupregulates the expression of downstream genes that are involvedin lipid biosynthesis. This elevation in SREBP processing andactivation is not caused by mislocalization of S1P or S2P (alsoknown as MBTPS1 and MBTPS2, respectively), two Golgi-residentendoproteases that are involved in SREBP processing, but insteadby increased Golgi residence of SREBPs, leading to their increasedsusceptibility to processing by the endoproteases. Analyses usinga processing-defective SREBP mutant suggest that a fraction ofSREBP molecules undergo basal cycling between the ER and Golgiin complex with SREBP cleavage-activating protein (SCAP).Furthermore, we show that SCAP alone is retrieved from the Golgiand moves to the ER after processing of SREBP under sterol-deficient conditions. Thus, our observations indicate that COPI-mediated retrograde trafficking is crucial for preventing unnecessarySREBP activation, by retrieving the small amounts of SCAP–SREBPcomplex that escape from the sterol-regulated ER retentionmachinery, as well as for the reuse of SCAP.

KEY WORDS: COPI-coated vesicles, Golgi complex, Lipogenesis,SCAP, SREBP

INTRODUCTIONIn eukaryotic cells, triacylglycerols (TAGs) and steryl esters arestored in cytoplasmic lipid droplets. According to the prevailingview of lipid droplet biogenesis, TAGs and steryl esters accumulatebetween the two leaflets of the endoplasmic reticulum (ER)membrane and gradually grow into globules. The nascent lipiddroplets then bud into the cytoplasm to generate isolated lipiddroplets (Ohsaki et al., 2014; Wilfling et al., 2014a). By contrast,neutral lipids in lipid droplets are mobilized through the actionof lipases, including adipose triglyceride lipase (ATGL, also knownas PNPLA2). Thus, lipid storage in lipid droplets is in dynamicequilibrium between the generation and mobilization of lipids.Systematic studies using small interfering (si)RNA screening

have revealed that knockdown of components of COPI-mediated

Golgi-to-ER retrograde trafficking, such as COPI subunits, ArfGTPases and the Arf-guanine nucleotide exchange factor GBF1,gives rise to an increase in lipid droplet size (Beller et al., 2008; Guoet al., 2008). These data suggest that COPI-mediated traffickingdirectly regulates lipid storage and/or mobilization. Furthermore,COPI-dependent delivery of ATGL to lipid droplets has beenimplicated in lipolysis, suggesting that inhibition of COPI-mediatedtrafficking results in lipid storage (Ellong et al., 2011; Soni et al.,2009). We have recently confirmed that knockdown of COPI-mediated trafficking components results in increased lipid storage;however, we failed to show direct evidence for the involvement ofCOPI-mediated trafficking in the delivery of ATGL to lipid droplets(Takashima et al., 2011). Therefore, we were interested inlipogenesis as well as lipolysis in order to understand the role ofCOPI-mediated trafficking in lipid homeostasis.

Sterol regulatory element-binding proteins (SREBPs) aretranscription factors that function as master regulators of genesthat are responsible for the synthesis of fatty acids and cholesterol(Goldstein et al., 2006; Horton et al., 2002). SREBP1 and SREBP2(also known as SREBF1 and SREBF2, respectively) are synthesizedas transmembrane precursors; there are two SREBP1 variants,designated SREBP1a and SREBP1c, which are derived from asingle gene through the use of alternative transcription start sites.Under sterol-rich conditions, they are retained in the ER in complexwith an escort protein, SREBP cleavage-activating protein (SCAP),which in turn forms a complex with the ER-resident Insig proteins(of which there are two, Insig1 and Insig 2) (see Fig. 6A). Whencellular sterol is depleted, SCAP undergoes a conformationalchange, leading to its release from Insig proteins. Liberated SCAP,which can interact with the COPII coat proteins, escorts SREBPsfrom the ER to the Golgi complex through COPII-coated vesicles.At the Golgi, SREBPs undergo sequential limited proteolysis bysite-1 and site-2 proteases (S1P and S2P; also known as MBTPS1and MBTPS2, respectively), thereby releasing the SREBPtranscription factor domain (TFD) from the N-terminal cytosolicregion. The TFD is then translocated into the nucleus and promotesthe expression of various genes that are involved in lipidbiosynthesis (see Fig. 6B). According to the prevailing model(Goldstein et al., 2006), the SCAP–SREBP complex is retained inthe ER under sterol-rich conditions, preventing unnecessary SREBPactivation (see Fig. 6A).

In this study, we found that inhibition of COPI-mediatedretrograde trafficking promotes the processing of SREBPs to yieldthe mature TFDs, which are then translocated into the nucleus toupregulate the expression of downstream genes that are involved inlipid biosynthesis. More importantly, the data indicate that althougha fraction of the SCAP–SREBP complex constitutively escapes thesterol-regulated ER-retention machinery, the proteins that haveescaped are retrieved from the Golgi, back to the ER, through COPI-mediated trafficking. Furthermore, we found that SCAP aloneReceived 1 October 2014; Accepted 12 June 2015

1Department of Physiological Chemistry, Graduate School of PharmaceuticalSciences, Kyoto University, Kyoto 606-8501, Japan. 2Department of AppliedBiological Chemistry, Graduate School of Agricultural and Life Sciences, Universityof Tokyo, Tokyo 113-8657, Japan. 3Career-Path Promotion Unit for Young LifeScientists, Kyoto University, Kyoto 606-8501, Japan.

*Author for correspondence (kazunaka@pharm.kyoto-u.ac.jp)

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undergoes COPI-mediated retrieval from the Golgi after regulatedER-to-Golgi trafficking of the SCAP–SREBP complex, andfollowing SREBP processing.

RESULTSA block in COPI-mediated trafficking increases nuclearSREBPaccumulation and theexpressionof genes involved inlipid biosynthesisWe have previously shown that cells depleted of any component ofCOPI-mediated trafficking, including β-COP (also known asCOPB1), exhibit enlarged lipid droplets associated with anincrease in cellular TAG content (Takashima et al., 2011); COPI-coated vesicles are involved in retrograde protein trafficking fromthe cis-Golgi to the ER, whereas COPII-coated vesicles are involvedin anterograde trafficking of proteins, including the transport of theSCAP–SREBP complex, from the ER to the Golgi (Harter andWieland, 1996). This prompted us to examine whether SREBPs,which are master regulators of TAG and cholesterol biosynthesis,

are implicated in the observed increase in cellular lipid storage, forthe following reasons – (i) ER-to-Golgi trafficking regulates theactivation of SREBPs; under sterol-depleted conditions, SCAPescorts SREBPs to the Golgi through COPII-coated vesicles, and atthe Golgi, SREBPs undergo sequential processing by the Golgi-resident S1P and S2P endoproteases to liberate the cytosolic TFDs(Goldstein et al., 2006; Horton et al., 2002) (also see Fig. 6B). (ii)Inhibition of COPI-mediated trafficking often perturbs the dynamicequilibria of proteins between the ER and the Golgi (for example,see Saitoh et al., 2009), and can therefore perturb the subcellulardistribution of SREBPs and/or the endoprotease(s).

To determine whether SREBPs are activated under COPI-deficient conditions, we treated HeLa cells with control smallinterfering (si)RNAs or siRNAs against β-COP (Fig. 1A,B), asdescribed previously (Saitoh et al., 2009; Takashima et al., 2011).We then incubated these cells in medium containing lipoprotein-deficient serum (LPDS) and oleic acid that had been supplementedwith or without 25-hydroxycholesterol (25-HC), which blocks the

Fig. 1. β-COP knockdown increases nuclear SREBP accumulation and the expression of genes involved in lipogenesis. (A–D,H,I) HeLa cells that hadbeen cultured in normal medium were transfected with control siRNAs (for LacZ, siControl) or siRNAs against β-COP (siβ-COP) and incubated for 20 h.(A) Lysates prepared from the siRNA-transfected cells were processed for immunoblot analysis with an antibody against β-COP (left) or β-tubulin as a control(right). (B) The siRNA-transfected cells were co-immunostained for β-COP (a,b) and GM130 as a Golgi marker (a′,b′). (C,D,H,I) After switching the medium toMEM supplemented with 10% LPDS and 400 µM oleic acid, with and without 1 µg/ml 25-HC (+ and – sterol, respectively), the siRNA-transfected cells wereincubated for 16 h and processed for staining with BODIPY 493/503 (C), TAG quantification (D), or RT-qPCR analyses for SCD (H) or HMGCR (I). (E) After themedium switching, the siRNA-transfected cells were incubated for 6 h, and after addition of ALLN (a final concentration, 100 μM) to inhibit protein degradation bythe proteasome, were further incubated for 6 h. The cells were then fractionated into nuclear and cytoplasmic fractions, which were processed for immunoblotanalysis for SREBP1, SREBP2, TBP (a nuclear protein) or calnexin (an ER protein). (F,G) Cells were transfected with a reporter construct for the ACC1 (F) orSQS (G) promoter, transfected with siControl or siβ-COP and cultured in MEM supplemented with 10% LPDS and 400 µM oleic acid, with or without 1 µg/ml25-HC. The cells were then subjected to luciferase assays. (D,F–I) The data are means±s.d. of three independent experiments. P-values were calculated usingone-way ANOVA followed by Tukey’s test. Scale bars: 10 μm (B,C).

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exit of SREBPs from the ER and thereby suppresses their processingand activation at the Golgi. The cells were then stained withBODIPY 493/503 in order to visualize lipid droplets, assayed forcellular TAG content or subjected to fractionation to separate thenuclear and cytoplasmic (containing the ER) fractions, which werethen processed for immunoblot analysis of SREBP1 or SREBP2. Asshown in Fig. 1C, lipid deposition in lipid droplets was elevated inthe β-COP-knockdown cells (Fig. 1Cc,Cd) relative to control cells(Fig. 1Ca,Cb) irrespective of the presence (Fig. 1Ca,Cc) or absence(Fig. 1Cb,Cd) of sterol. Almost in parallel with the increased lipiddeposition, the cellular TAG content was significantly increased byβ-COP knockdown, again irrespective of the presence or absence ofsterol (Fig. 1D).Under sterol-depleted conditions, SREBPs undergo COPII-

mediated anterograde trafficking from the ER to the Golgi, wherethey are processed by S1P and S2P to liberate their cytosolic TFDs,which are then translocated into the nucleus (Goldstein et al., 2006;Horton et al., 2002). As shown in Fig. 1E, the nuclear mature formsof SREBP1 and SREBP2 were barely detectable in control cells thathad been incubated in the presence of sterol (lane 1), whereassubstantial amounts of the mature forms were found in the nuclearfraction of control cells that had been incubated in the absence ofsterol (lane 3).When β-COP-knockdown cells were incubated in theabsence of sterol, the levels of the nuclear mature forms of SREBP1and SREBP2 were slightly higher than those in control cells that hadbeen incubated in the absence of sterol (compare lane 4 with lane 3).The most striking changes were observed in β-COP-knockdowncells that had been incubated with sterol (compare lane 2 withlane 1) – substantial amounts of the mature forms of SREBP1 andSREBP2 were found in the nuclear fractions, even in the presence ofsterol. Thus, processing of SREBPs to yield the mature TFDs wasenhanced by knockdown of β-COP, irrespective of the cellularsterol level.The results described above implicate SREBP activation in the

observed increases in lipid deposition and TAG content in β-COP-knockdown cells. To further test this hypothesis, cells weretransfected with a luciferase reporter plasmid containing thepromoter region of the gene encoding acetyl-CoA carboxylase 1(ACC1, also known as ACACA) or squalene synthase (SQS, alsoknown as FDFT1), which are primarily controlled by SREBP1 andSREBP2, respectively (Horton et al., 2002). The transfected cellswere treated with siRNAs against β-COP and subjected to luciferaseassays to assess the transcriptional activity of endogenous SREBPs.Under sterol-depleted conditions, the activity of both the ACC1 andSQS promoters was approximately doubled by knockdown of β-COP(Fig. 1F,G). Furthermore, knockdown of β-COP increased theactivity of both promoters even in the presence of sterol, although thebasal activities were lower than those in the absence of sterol.We also examined the effects of β-COP knockdown on the levels

of the mRNAs encoding stearoyl-CoA desaturase (SCD, also knownas SCD5) and HMG-CoA reductase (HMGCR), which are regulatedmainly by SREBP1 and SREBP2, respectively (Horton et al.,2002), by using quantitative reverse-transcription PCR (RT-qPCR)analyses. As shown in Fig. 1H,I, the results were generally consistentwith those obtained using the reporter assay – i.e. β-COP knockdownresulted in an increase in the levels of both mRNAs under bothsterol-rich and sterol-depleted conditions – although the relativeexpression levels were lower under the sterol-rich conditions. Thus,both the reporter assay and RT-qPCR analyses revealed that theactivity of SREBP1 and SREBP2 is increased by knocking downβ-COP, even under sterol-rich conditions, under which the exit ofSCAP–SREBP from the ER is normally suppressed.

Localization of S1P or S2P is not altered in COPI-compromised cellsTo explore the mechanism underlying the increased activation ofSREBPs under COPI-compromised conditions, we first addressedthe possibility thatGolgi-to-ER retrograde trafficking of the processingendoproteases S1P and S2P is elevated. This hypothesis was basedon the fact that the treatment of cells with brefeldin A (BFA), whichcauses the Golgi complex to collapse into the ER by inhibiting Arfguanine nucleotide exchange factors (Jackson, 2000; Sciaky et al.,1997), results in increased processing of SREBP2 owing tomislocalization of S1P to the ER (DeBose-Boyd et al., 1999), aswell as in increased lipid deposition and TAG content (Beller et al.,2008; Takashima et al., 2011). As shown in supplementary materialFig. S1, exogenously expressed hemagglutinin (HA)-tagged S1Pand S2P were localized predominantly in the perinuclear Golgiregion in control cells (supplementary material Fig. S1Aa–a‴,Ba–a‴), as revealed by staining for the Golgi protein giantin (alsoknown as GOLGB1). Consistent with a previous report (Bartz et al.,2008; DeBose-Boyd et al., 1999), treatment with BFA altered thestaining pattern of S1P or S2P so that it matched that of the ER(supplementary material Fig. S1Ac–c‴,Bc–c‴). In cells treatedwith BFA or golgicide A, which is a more specific Arf guaninenucleotide exchange factor inhibitor, processing of SREBP1c wasobserved even in the presence of sterol (supplementary materialFig. S1C), as reported for SREBP2 in BFA-treated cells (DeBose-Boyd et al., 1999). In contrast to the effect of BFA, β-COPknockdown (supplementary material Fig. S1Ab–b‴,Bb–b‴) didnot alter the Golgi localization of S1P, S2P or giantin. Theseobservations are consistent with our previous study showingthat β-COP knockdown does not affect the localization of otherGolgi proteins (GM130 and golgin-97; Saitoh et al., 2009) andindicate that, in COPI-compromised cells, a mechanism otherthan redistribution of S1P and/or S2P contributes to SREBPactivation; however, they do not completely exclude the possibilitythat a small fraction of S1P and/or S2Pmolecules are redistributed tothe ER.

COPI-dependent retrieval of SCAP and SREBPs from theGolgi to the ERIn light of the observations described above, we investigatedwhether SREBP trafficking is altered in COPI-compromised cells.To this end, we exploited a SREBP1c mutant (R503A) that isdefective in S1P-catalyzed cleavage (Hua et al., 1996b) andcompared the subcellular localizations of wild-type (WT) andmutant SREBP1c proteins in control and β-COP-knockdown cells.The presence of exogenously expressed SREBP in large excess ofthe endogenous level of the escort protein SCAP prevents theexpressed SREBP from exiting the ER, even under sterol-depletedconditions (Hua et al., 1996a; also see supplementary materialFig. S3A); to circumvent this problem, we co-infected HeLa cellswith recombinant retroviruses encoding red fluorescent protein(mRFP)-tagged SREBP1c and SCAP–HA in order to achievemoderate expression levels of both proteins (Fig. 2).

In the control cells, under both sterol-rich (Fig. 2Aa–Aa‴,Ba) andsterol-depleted (Fig. 2Ab–Ab‴,Bb) conditions, mRFP–SREBP1c(WT) was primarily found in the nucleus (Fig. 2Aa,Ba, and Ab,Bb,respectively), whereas SCAP–HAwas mainly localized in the Golgi(Fig. 2Aa′,Ab′). This observation indicates that exogenouslyexpressed SREBP1c and SCAP are constitutively trafficked to theGolgi, and that the former is subsequently cleaved by the S1P andS2P endoproteases and translocated into the nucleus. This result isin agreement with previous studies showing that overexpression of

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SCAP leads to a marked increase in the level of the nuclear matureSREBP2, even under sterol-rich conditions (Hua et al., 1996a; Yanget al., 2002), because overexpressed SCAP saturates the endogenouslevel of Insig proteins, and excess free SCAP escorts SREBPs to theGolgi, irrespective of the cellular sterol level.WhenmRFP–SREBP1c(R503A) and SCAP–HAwere co-expressed,

both the SREBP1c mutant and SCAP were predominantly foundin the ER under both sterol-rich (Fig. 2Ca–Ca‴,Da) and sterol-depleted (Fig. 2Cb–Cb‴,Db) conditions. These observations weresomewhat unexpected given the results that have been describedabove (Fig. 2A,B) and in previous studies (Hua et al., 1996a; Yanget al., 2002), which indicate that exogenously expressed SREBPsand SCAP constitutively exit the ER. Two models might explain theobservations obtained by co-expressing SREBP1c(R503A) andSCAP – (i) the complex of SCAP and SREBP1c(R503A) could notexit the ER; (ii) although the SCAP–SREBP1c(R503A) complexwas constitutively transported from the ER to the Golgi, thecomplex was efficiently retrieved back to the ER. The results of thefollowing experiments support the latter possibility.To achieve sterol-regulated transport of the SCAP–SREBP

complex, we then triple-infected cells with recombinantretroviruses encoding mRFP–SREBP1c, SCAP–HA and Insig2–FLAG (Fig. 3). In contrast to cells that lacked exogenous Insig2expression, in which SREBP1c(WT) and SCAP constitutivelyexited the ER (Fig. 2A,B), cells co-expressing Insig2 exhibitedregulated changes in the localization of SREBP1c and SCAP –under sterol-rich conditions (Fig. 3Aa–Aa‴,Ba), SREBP1c(WT)and SCAP were mainly found in the ER (Fig. 3Aa,Aa′), and theformer was barely detectable in the nucleus (Fig. 3Aa), whereasunder sterol-depleted conditions (Fig. 3Ac–Ac‴,Bc), SREBP1c(WT) and SCAP were found primarily in the nucleus (Fig. 3Ac) andthe Golgi (Fig. 3Ac′), respectively. When the triple-infected cellswere subjected to knockdown of β-COP, under either sterol-depleted (Fig. 3Ad–Ad‴,Bd) or sterol-rich (Fig. 3Ab–Ab‴,Bb)conditions, SREBP1c(WT) and SCAP were localizedpredominantly in the nucleus (Fig. 3Ab,Ad) and the Golgi(Fig. 3Ab′,Ad′), respectively. Comparison of the localization ofSREBP1c(WT) and SCAP in control cells (Fig. 3A,Ca–Ca‴), and inβ-COP-knockdown cells (Fig. 3A,Cb–Cb‴) under sterol-richconditions revealed that a block in COPI-mediated transportcaused an accumulation of SCAP–SREBP and increasedSREBP1c processing in the Golgi. It is noteworthy that, in β-

COP-knockdown cells, the Golgi structure revealed by staining ofgiantin did not undergo any substantial changes compared with thatof control cells, nor did giantin redistribute to the ER (Fig. 3Ab″,Ad″),consistent with our previous report showing that β-COPknockdown does not affect the localization of other Golgiproteins (Saitoh et al., 2009). A possible explanation for theseobservations is that, even under sterol-rich conditions, a fraction ofthe SCAP–SREBP complex traffics to the Golgi complex throughCOPII-coated vesicles (Espenshade et al., 2002; Goldstein et al.,2006), but that the escaped complex is returned to the ER throughCOPI-coated vesicles.

To demonstrate basal cycling of the SCAP–SREBP complexbetween the ER and the Golgi, we exploited the processing-defective SREBP1c(R503A) mutant (Fig. 3C,D). In control cellsthat had been subjected to triple infection with retroviruses formRFP–SREBP1c(R503A), SCAP–HA and Insig2–FLAG, bothSCAP (Fig. 3Ca′,Cc′) and SREBP1c(R503A) (Fig. 3Ca,Cc) weremainly localized in the ER under both sterol-rich (Fig. 3Ca–Ca‴,Da) and sterol-depleted (Fig. 3Cc–Cc‴,Dc) conditions. In strikingcontrast to the control cells, both SREBP1c(R503A) (Fig. 3Cb,Cd,Db,Dd) and SCAP (Fig. 3Cb′,Cd′) were found predominantly in theGolgi, both under sterol-rich (Fig. 3Cb–Cb‴) and sterol-depleted(Fig. 3Cd–Cd‴) conditions in the β-COP-knockdown cells. Toexclude the possibility that, in sterol-depleted control cellsexpressing mRFP–SREBP1c(R503A), the absence of SCAP andSREBP1c(R503A) in the Golgi was due to degradation of theseproteins (see Discussion), and that the presence of these proteins inthe ER reflected their de novo synthesis, cells that expressed eitherSREBP1c(WT) or SREBP1c(R503A), as well as SCAP and Insig2were incubated in the presence of cycloheximide (CHX) to block denovo protein synthesis, and we examined the protein levels. Asshown in supplementary material Fig. S2, neither the SREBP1c norSCAP level was substantially different between cells expressingSREBP1c(WT) or SREBP1c(R503A) (supplementary materialFig. S2, lanes 1 and 3), although the SCAP level was slightlylower under sterol-depleted conditions than under sterol-richconditions (compare supplementary material Fig. S2, lane 2 vslane 1 and lane 4 vs lane 3). Taken together, the data shown in Fig. 3and supplementary material Fig. S2 indicate that, even under sterol-rich conditions, a fraction of the SCAP–SREBP complex leaks outof the ER into the Golgi, but the complex is returned to the ERthrough COPI-coated vesicles (see Fig. 6C).

Fig. 2. Constitutive exit of exogenouslyexpressed SREBPand SCAP from the ER.HeLacells were co-infected with recombinantretroviruses for either mRFP–SREBP1c(WT)(A,B) or –SREBP1c(R503A) (C,D) and SCAP–HA,and then cultured in MEM supplemented with 10%LPDS and 400 µM oleic acid in the presence(+ sterol, a–a‴) or absence (− sterol, b–b‴) of1 µg/ml 25-HC. The cells were then stained for HA(a′,b′) and giantin (a″,b″). Cells with typical nuclearfluorescence of mRFP–SREBP1c(WT) (A) or Golgifluorescence of mRFP–SREBP1c(R503A)(C) were counted, and the percentages wereexpressed as bar graphs (B,D, respectively).Scale bar: 10 μm.

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Suppression of SREBP activation reverses the increase inTAG content that is induced by COPI knockdownIf a basal fraction of SCAP–SREBP escapes the ER retentionmachinery, we predicted that depletion of SCAP would blockthe export of SREBPs and thereby limit its accessibility to theGolgi-resident endoproteases. This prediction is supported by theobservation that when cells overexpressing the wild type or R503Amutant of mRFP-SREBP1c alone were treated with siRNAs againstβ-COP, both SREBP1c constructs were mainly found in the ER(supplementary material Fig. S3); even in β-COP-knockdown cells,SREBP1c(WT) was never found in the nucleus (supplementarymaterial Fig. S3B) and SREBP1c(R503A) was never found in theGolgi (Fig. S3D).

We therefore knocked down SCAP in addition to β-COP; theknockdown efficiencies were examined by immunoblotting(supplementary material Fig. S4). As shown in Fig. 4,simultaneous knockdown of SCAP substantially suppressed theincrease in the levels of the nuclear mature forms of endogenousSREBPs (Fig. 4A, compare lane 2 vs lane 4 and lane 6 vs lane 8) andsuppressed the increase in the expression levels of the reporter genes(Fig. 4B,C) induced by β-COP knockdown, confirming that theincreased activation of SREBPs requires prior export from the ER ofSREBPs, escorted by SCAP.

Moreover, if this aberrant activation of SREBPs contributes tothe increase in TAG content that is induced by β-COPknockdown, inhibition of SREBP activation by simultaneous

Fig. 3. COPI-dependent recycling of SREBP and SCAP to the ER. Cells were triple-infected with retroviruses for either mRFP–SREBP1c (WT) (A,B) or–SREBP1c(R503A) (C,D), SCAP–HAand Insig2–FLAG; transfectedwith control siRNAs (siRNAs against LacZ, siControl; a–a‴,c–c‴) or siRNAsagainst β-COP (siβ-COP; b–b‴,d–d‴); cultured in MEM supplemented with 10% LPDS and 400 µM oleic acid, with (+ sterol, a–a‴,b–b‴) or without (− sterol, c–c‴,d–d‴) 1 µg/ml 25-HC;and then stained for HA (a′–d′) and giantin (a″–d″). The cells with typical nuclear fluorescence of mRFP–SREBP1c(WT) (A) and those with Golgi-localizedfluorescence of mRFP–SREBP1c(R503A) (C) were counted, and the percentages were expressed as bar graphs (B and D, respectively). Scale bar: 10 μm.

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knockdown of SCAP and β-COP should suppress the increase. Infact, in parallel with the levels of mature SREBPs, the increase inthe cellular TAG content induced by β-COP knockdown weresignificantly reversed by simultaneous knockdown of SCAP(Fig. 4D). Thus, the increase in TAG content that is induced bythe block in COPI-mediated retrograde transport depends on theaberrant activation of SREBPs.

Retrograde trafficking of SCAP after processing andactivation of SREBPLittle information is available for the fate of SCAP after processingand activation of SREBP at the Golgi, although a previous cell-fractionation study reports that, even under low-sterol conditions,most SCAP appears to be present in the ER fraction (Nohturfft et al.,1999). To unequivocally determine whether SCAP that has beenliberated from SREBP at the Golgi returns to the ER, we adopted thefollowing protocol – HeLa cells that had been infected withrecombinant retrovirus vectors encoding mRFP–SREBP1c, SCAP–HA and Insig2–FLAG were cultured in minimum essential medium(MEM) that had been supplemented with fetal bovine serum (FBS)and were then incubated for 90 min in MEM containing LPDS,2-hydroxypropyl β-cyclodextrin (HPCD) and CHX in order tothoroughly deplete cellular sterols and block de novo proteinsynthesis. After washing twice with MEM containing LPDS andCHX, the cells were incubated inMEM containing FBS, 25-HC andCHX for 30 min to replenish cellular sterol. Before steroldeprivation, both SCAP and SREBP1c were predominantlylocalized in the ER (Fig. 5Aa,Aa′). After sterol deprivation for90 min, a major fraction of SCAP was found in the Golgi, whereasSREBP1c was predominantly found in the nucleus, indicative of itsprocessing at the Golgi and subsequent translocation into the

nucleus (Fig. 5Ab). Thus, the SCAP–SREBP complex appeared tobe trafficked from the ER to the Golgi in response to a decrease inthe cellular sterol level. After sterol replenishment for 30 min, theGolgi localization of SCAP was completely lost and only its ERlocalization was evident (Fig. 5Ac′). Immunoblot analysis of celllysates indicated that the protein level of SCAPwas not substantiallychanged during the period of sterol depletion and re-addition(Fig. 5B), excluding the possibility that the SCAP protein in theGolgi undergoes degradation after sterol re-addition. The mostplausible explanation for these observations is that SCAPmolecules, once trafficked to the Golgi under sterol-depletedconditions, recycle back to the ER upon sterol replenishment.Taking into account the fact that SCAP accumulates in the Golgi –even under sterol-rich conditions – in β-COP-knockdown cells(Fig. 3Cb′), SCAP is most likely recycled back to the ER by COPI-mediated trafficking.

We also followed sterol-dependent changes in the localization ofSCAP by using time-lapse recording. Cells expressing mRFP–SREBP1c, enhanced green fluorescent protein (EGFP)-taggedSCAP and Insig2–FLAG were incubated for 90 min in MEMcontaining LPDS, HPCD and CHX in order to deplete the cellularsterols and block de novo protein synthesis. After washing twicewith MEM containing LPDS and CHX, the cells were subjectedto time-lapse recording in MEM containing FBS, 25-HC andCHX (+sterol) or that containing LPDS, HPCD and CHX (−sterol).At time=0, SCAP–EGFP exhibited Golgi-localization, whereasmRFP–SREBP1c exhibited nuclear localization (Fig. 5C,D, leftpanels). Under continually sterol-deprived conditions, thefluorescence signals of SCAP–EGFP at the Golgi did not changesubstantially, although both the signals at the ER and Golgigradually decreased, probably owing to basal levels of degradation

Fig. 4. SCAP knockdown attenuatesSREBP activation, and β-COPknockdown increases TAG content.(A,D) Cells were transfected with controlsiRNAs (siControl), siRNAs targetingβ-COP (siβ-COP) or SCAP (siSCAP), or acombination of siRNAs against β-COPand SCAP; they were then cultured inMEM supplemented with 10% LPDSand 400 µM oleic acid, with and without1 µg/ml 25-HC (+ and – sterol,respectively). The cells were subjected toanalysis of SREBP processing (A) orTAG quantification (D). (B,C) Cells weretransfected with the ACC1 (B) or SQS (C)reporter construct; transfected withcontrol siRNAs, siRNAs targeting β-COPor SCAP, or a combination of siRNAstargeting β-COP and SCAP; cultured inMEM supplemented with 10% LPDS and400 µM oleic acid, with or without 1 µg/ml25-HC; and then subjected to luciferaseactivity assays. (B–D) The data aremeans±s.d. of three independentexperiments. P-values were calculatedusing one-way ANOVA followed byTukey’s test.

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of SCAP–EGFP and/or photobleaching of the EGFP moiety in theabsence of its de novo synthesis (Fig. 5C,E, dotted lines;supplementary material Movie 1). In striking contrast, when themedium was switched to one that was sterol-rich, SCAP–EGFPin the Golgi region was virtually undetectable within 10 min(Fig. 5D,E, green solid line; supplementary material Movie 2).We failed to observe a significant increase in the SCAP–EGFPsignal at the ER that correlated with a marked decrease in its signal inthe Golgi region; this was attributable, at least in part, to the fact thatthe surface area of the ER was estimated to be at least fivefold largerthan that of the Golgi (Griffiths et al., 1989; Sciaky et al., 1997).However, the signals at the ER after sterol replenishment (Fig. 5E,red solid line) were significantly higher than those in the continual

absence of sterol (red dotted line). Taking into account that the SCAPprotein level did not change during the period of sterol depletion andre-addition (Fig. 5B), these observations indicate that SCAP–EGFPmolecules, once delivered to the Golgi, were recycled back to the ER,concomitant with the recovery of the cellular sterol level.

DISCUSSIONPrevious studies by our group and others have shown that a block inGolgi-to-ER retrograde trafficking through COPI-coated vesiclescauses an increase in lipid deposition (Beller et al., 2008; Guo et al.,2008; Soni et al., 2009; Takashima et al., 2011). Here, we extendedour previous study and showed that aberrant activation of SREBPs,master regulators of biosynthesis of fatty acids and cholesterol, can

Fig. 5. SCAP recycling to the ER after processing of SREBP at the Golgi. (A,B) Cells were triple-infected with retroviruses encoding mRFP–SREBP1c,SCAP–HA and Insig2–FLAG, and cultured inMEM supplemented with 10%FBS (Aa-a‴; B, lane 1). The cells were then incubated inMEM containing 10% LPDS,1% HPCD and 50 µg/ml CHX for 90 min to deplete cellular sterols and inhibit de novo protein synthesis (Ab-b‴; B, lane 2). After washing twice with MEMcontaining LPDS and CHX, the cells were incubated with MEM containing FBS, 1 µg/ml 25-HC and CHX for 30 min (Ac-c‴; B, lane 3). The cells were stained forHA (Aa′–Ac′) and giantin (Aa″–Ac″), or processed for immunoblot analysis with an antibody against RFP (upper panel) or HA (lower panel). After sterol depletion,a considerable fraction of SCAP–HA became localized in the Golgi region (Ab′), and after sterol re-addition, the Golgi signals for SCAP–HA were no longerdetectable, and only ER signals were prominent (Ac′). (C,D) Cells that had been triple-infected with retroviruses encoding mRFP–SREBP1c, SCAP–EGFP andInsig2–FLAG were depleted of cellular sterols and washed as in A,B. The cells were then incubated in MEM containing LPDS and CHX (C, − sterol) or in thatcontaining FBS, 25-HC and CHX (D, + sterol) and subjected to time-lapse recording. Images were acquired every 5 min. (E) The total intensities of the EGFPsignal in the Golgi region (surrounded by green lines) and in the ER (the rest of the cell area) were estimated using NIS-elements (Nikon). The results areexpressed as percentages of signals remaining in theGolgi region (green lines) and in the ER (red lines) in the cells incubatedwith sterol-deficient (− sterol, dottedlines) or -replenished medium (+ sterol, solid lines) at the indicated time points. The data are means±s.d. from five independent cells. *P<0.05; **P<0.02;***P<0.005; Student’s t-test.

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be ascribed, at least in part, to the increased lipid deposition thatoccurs under COPI-compromised conditions.The current conventional model for regulated activation of

SREBPs, followed by the stimulation of biosynthesis of fatty acidsand cholesterol, was proposed by Brown, Goldstein and colleaguesbased on a series of elegant studies (Goldstein et al., 2006; Hortonet al., 2002) (see Fig. 6A,B). In this model, when cholesterol buildsup in ER membranes, the sterol causes SCAP to adopt aconformation that forces its binding to Insig proteins and prevents

its binding to the COPII coat proteins. Consequently, the SCAP–SREBP complex fails to exit the ER (Fig. 6A). When cells aredepleted of sterol, SCAP dissociates readily from Insig proteins andcan bind to the COPII coat proteins, and the SCAP–SREBPcomplex is then transported towards the Golgi complex (Fig. 6B).

One important point to be stressed about this conventional modelis that the SCAP–SREBP complex is not assumed to escape thesterol-regulated ER retention machinery. However, it is known that anumber of ER proteins have ER ‘retention signals’ that actually

Fig. 6. Models for SCAP–SREBP trafficking and SREBP activation. In the conventional model (A,B), (A) the SCAP–SREBP complex is retained in the ER byinteracting with Insig proteins under high-sterol conditions, or (B) the complex is exported from the ER through COPII-coated vesicles, and SREBP is processedby S1P and S2P at the Golgi and translocated into the nucleus under low sterol-conditions. In the revised model based on the present study (C,D), (C) a basalfraction of the SCAP–SREBP complex leaks out of the ER even under high-sterol conditions but is retrieved from the Golgi to the ER through COPI-coatedvesicles before SREBP can be processed by S1P and S2P. (D) Under low-sterol conditions, the proportion of the SCAP–SREBP complex exiting the ER isincreased, and a substantial fraction of the SREBP molecules that arrive at the Golgi are processed by S1P and S2P, and are translocated into the nucleus,whereas SCAP molecules are recycled to the ER through COPI-coated vesicles for reuse. (A,D) Cholesterol is represented by a schematic steroid backbone.

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serve as ‘retrieval signals’ from the Golgi in the regulated ER–Golgirecycling system (Harter and Wieland, 1996). For example, mostsoluble ‘ER-resident proteins’ have a C-terminal KDEL sequencethat serves as a retrieval signal by binding to the KDEL receptor atthe Golgi. Some ER transmembrane proteins have a C-terminalKKxx/KxKxx sequence that serves as a binding site for the COPIcoat proteins, and another population of ER transmembrane proteinsare recycled back to the ER by binding to the Rer1 protein at theGolgi. An early study of Brown, Goldstein and colleagues indirectlysupports the view that a small fraction of the SCAP–SREBP complexcan exit the ER even under sterol-rich conditions; they report that,even under sterol-rich conditions, a proportion of the N-linkedoligosaccharide chains attached to SCAP are partially resistant todigestion by endoglycosidase H, although a high proportion of themare sensitive to the glycosidase (Nohturfft et al., 1998).We first noticed that a fraction of SCAP–SREBP undergoes basal

cycling between the ER and the Golgi in cells that exogenouslyco-expressed SREBP1c(R503A) and SCAP (Fig. 2C,D). In theabsence of exogenous expression of Insig proteins, SCAP–SREBPconstitutively exits the ER and traffics towards the Golgi,irrespective of the cellular sterol level (Goldstein et al., 2006; Huaet al., 1996a; Yang et al., 2002), as observed in cells thatco-expressed SREBP1c(WT) and SCAP (Fig. 2A,B). By contrast,both SREBP1c(R503A) and SCAP were predominantly found inthe ER under both sterol-rich and sterol-depleted conditions(Fig. 2C,D). Furthermore, essentially the same results wereobtained when SREBP1c(R503A), SCAP and Insig2 were co-expressed (Fig. 3Ca–Ca‴,Cc–Cc‴,Da,Dc).In striking contrast to the control cells, when cells that co-

expressed SREBP1c(R503A), SCAP and Insig2 were subjected toknockdown of β-COP, both the SREBP1c mutant and SCAP werepredominantly found in the Golgi complex under both sterol-rich(Fig. 3Cb–Cb‴,Db) and sterol-depleted (Fig. 3Cd–Cd‴,Dd)conditions. In addition, when cells that co-expressed SREBP1c(WT), SCAP and Insig2 were treated with control siRNAs orsiRNAs against β-COP, and then cultured in the presence of sterol,both SREBP1c(WT) and SCAP were primarily found in the ER inthe control cells (Fig. 3Aa–Aa‴,Ba), whereas SREBP1c(WT) andSCAP were found mainly in the nucleus and in the Golgi,respectively, in β-COP-knockdown cells (Fig. 3Ab–Ab‴,Bb).Taken together, these observations strongly suggest that, evenunder sterol-rich conditions, a fraction of the SCAP–SREBPcomplex escapes the sterol-regulated ER-retention machinery,which is then retrieved from the Golgi to the ER through COPI-coated vesicles, without the majority of SREBP moleculesundergoing processing by the Golgi-resident proteases S1P andS2P (Fig. 6C); however, it is currently unknown why the SCAP–SREBP complex can return to the ER before SREBP processing bythe endoproteases. One possible mechanism that preventsproteolytic processing of SREBPs despite their continual shuttlingis that a large fraction of the SCAP–SREBP complex is retrievedbefore the complex reaches a compartment in which the S1Pendoprotease is resident, as COPI-coated vesicles mediate retrievalof proteins not only from the cis-Golgi but also from the ER–Golgiintermediate compartment (Harter and Wieland, 1996; Lowe andKreis, 1998; Watson and Stephens, 2005).The model shown in Fig. 6C is compatible with the fact that, even

under sterol-rich conditions, N-linked oligosaccharide chains ofSCAP molecules are partially resistant to digestion withendoglycosidase H (Nohturfft et al., 1998). By contrast, underlow-sterol conditions, SCAP that has been liberated from SREBP atthe Golgi probably returns to the ER for reuse (Nohturfft et al.,

1999), and the basal cycling of the SCAP–SREBP complex is likelyto continue (Fig. 6D).

What determines the COPI-mediated retrograde trafficking ofthe SCAP–SREBP complex? Neither SREBP nor SCAP has acanonical KKxx/KxKxx sequence, and our preliminary analysisfailed to show that knockdown of Rer1 affects the localization ofSREBP and SCAP. This question should be addressed in futurestudies.

While this current study was in progress, Shao and Espenshadehave suggested that uncleaved SREBP inhibits SCAP recyclingfrom the Golgi to the ER, and that instead, SCAP escorts SREBP tolysosomes for degradation, although the authors do not present anydata on the subcellular localization of SREBP or SCAP (Shao andEspenshade, 2014). By contrast, we show here that SREBP1c(R503A) – a mutant defective in S1P-catalyzed cleavage – andSCAP are primarily found in the ER under control conditions,whereas both accumulate in the Golgi when COPI-mediatedretrograde trafficking is inhibited (Fig. 3). Furthermore, the SCAPlevel in the presence of co-expressed SREBP1c(R503A) wascomparable to that in the presence of SREBP1c(WT) even whende novo protein synthesis was blocked (supplementary materialFig. S2; compare lane 1 vs lane 3 and lane 2 vs lane 4). Thus, ourresults unequivocally show that a large fraction of uncleavedSREBP molecules are recycled back to the ER, along with SCAP,through COPI-coated vesicles, although they do not exclude thepossibility that a small fraction of SREBP–SCAP undergoeslysosomal degradation. The apparent discrepancy between our dataand those of Shao and Espenshade might result from a difference inthe experimental systems used.We reached our conclusions by usingSREBP1c(R503A), which is not cleaved by S1P, whereas Shao andEspenshade have drawn their conclusions by using an S1P inhibitor(PF-429242) (Shao and Espenshade, 2014). Because S1P acts onmultiple substrates in addition to SREBPs, there could be otherfactor(s) that need to be processed by S1P activity to allow SCAP torecycle properly.Although it is beyond the scope of the current study,this possibility should be addressed in a future study.

In this study, we showed that activation of SREBPs is mainlyresponsible for increased levels of lipid deposition under COPI-depleted conditions. However, in view of the data that lipiddeposition, as revealed by using BODIPY 493/503 staining, wasmuch stronger in β-COP-knockdown cells under sterol-richconditions compared with that of control cells under sterol-deficient conditions (Fig. 1C) even though the nuclear SREBPlevels were comparable in these two situations (Fig. 1E), it ispossible that other mechanism(s) also underlie the increased lipiddeposition in COPI-depleted cells. For example, it has been reportedpreviously that COPI is required for the delivery of a lipolyticenzyme, ATGL, to lipid droplets (Soni et al., 2009). By contrast,other studies have shown that COPI can act directly on lipid dropletsto promote the budding of nano-droplets and, consequently, primethe targeting of proteins or membranes to lipid droplet surfaces(Thiam et al., 2013; Wilfling et al., 2014b). Nevertheless, our datapresented here unequivocally show that aberrant activation ofSREBP contributes, at least in part, to lipid deposition underCOPI-compromised conditions.

MATERIALS AND METHODSAntibodies and reagentsThe following antibodies were obtained from the indicated vendors –polyclonal rabbit anti-β-COP, Thermo Scientific; polyclonal rabbit anti-SCAP, Abgent; polyclonal rabbit anti-giantin, COVANCE; monoclonalmouse antibodies against SREBP1, β-tubulin and β-actin, Millipore;

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monoclonal mouse anti-SREBP2, MBL; monoclonal mouse antibodiesagainst calnexin and GM130, BD Biosciences; monoclonal mouse anti-TATA-binding protein (TBP), Santa Cruz Biotechnologies; polyclonalrabbit anti-RFP, MBL; monoclonal rat anti-HA, Roche AppliedScience; Alexa-Fluor-conjugated secondary antibodies, Molecular Probes;and horseradish-peroxidase-conjugated secondary antibodies, JacksonImmunoResearch Laboratories. BODIPY 493/503 was purchased fromMolecular Probes. BFA, 25-HC, CHX and oleic acid were from Sigma-Aldrich. ALLN was from Nacalai Tesque. HPCD was from Wako PureChemical Industries.

Recombinant plasmids and virusesExpression vectors for C-terminally HA-tagged S1P and N-terminallyHA-tagged S2P were constructed by subcloning a cDNA fragmentcontaining the entire coding sequence of human S1P or S2P into pcDNA3-HAC (Hosaka et al., 1996) or pcDNA4-HAN (Shinotsuka et al., 2002),respectively. The expression vector for C-terminally EGFP-tagged SCAPwas constructed by subcloning a cDNA fragment containing the entire codingsequence of human SCAP into pEGFP-N1. The expression vector for N-terminally mRFP-tagged SREBP1c was constructed by subcloning a cDNAfragment containing the entire coding sequence of human SREBP1c into thepcDNA3-Kozac-mRFP-C vector (Makyio et al., 2012). The R503Amutationof SREBP1cwas introduced into the SREBP1c cDNAusing the QuikChangeLightning Site-Directed Mutagenesis Kit (Agilent Technologies). Theexpression vector for C-terminally FLAG-tagged Insig2 was constructedby subcloning a cDNA fragment covering the entire coding sequence ofhuman Insig2 into pCAG-sk-FLAG-N3.Construction of pCMV5-SCAP-HAand Firefly luciferase reporter vectors, pGL4-ACC1 and pGVB2-SQS, havebeen described previously (Irisawa et al., 2009). pGL4.74(hRluc/TK), whichencodes Renilla luciferase, was purchased from Promega.

For production of mRFP–SREBP1c-encoding retrovirus, the mRFP–SREBP DNA fragment was amplified by using PCR of pcDNA3-mRFP-SREBP1c (described above), which was then inserted into the pMXs-purovector (a kind gift from Toshio Kitamura, The University of Tokyo, Tokyo,Japan) (Kitamura et al., 2003) using an In-Fusion HD Cloning kit(TaKaRa). For production of the retroviruses for SCAP–HA, SCAP–EGFP or Insig2–FLAG, the SCAP–HA, SCAP–EGFP or Insig2–FLAGDNA fragment was amplified from pCMV5-SCAP-HA, pEGFP-N1-SCAPor pCAG-Insig2-FLAG, and inserted into pMXs-bla or pMXs-neo. ThepMXs vector for mRFP–SREBP1c, SCAP–HA, SCAP–EGFP or Insig2–FLAGwas co-transfected with pEF-gag-pol and pCMV-VSVG-Rsv-Rev (akind gift from Hiroyuki Miyoshi, RIKEN BRC, Tsukuba, Japan) intoHEK293T cells to produce recombinant virus.

Plasmid transfection, virus infection and RNA interferenceβ-COP was knocked down using a pool of siRNAs, as described previously(Saitoh et al., 2009; Takashima et al., 2011). For knockdown of SCAP,a pool of siRNAs directed against nucleotides 1215–1725 (when the Anucleotide of the initiation methionine codon is assigned as residue 1) ofhuman SCAP mRNA was prepared using the BLOCK-iT RNAi TOPOtranscription kit (Invitrogen) and a PowerCut Dicer kit (Thermo Scientific).Cells were transfected for 24 h with the siRNA pool using Lipofectamine2000 (Invitrogen). The cells were then subcultured, incubated for a further24 h and re-transfected with the siRNA pool for 24 h. The cells were thensubcultured, incubated for 48 h and subjected to subsequent analyses.Plasmid transfection was performed using the X-tremeGENE 9 DNATransfection Reagents (Roche Applied Science). Cells were transfected withexpression plasmids 8 h before, or infected with recombinant retroviruses24 h before, transfection with siRNAs against β-COP.

Cell cultureHeLa cells were maintained in MEM (Sigma-Aldrich) supplemented with10% heat-inactivated FBS (Invitrogen). Before experiments, cells werecultured in MEM containing 10% LPDS (Sigma-Aldrich) and 400 μMoleicacid in the presence or absence of 1 μg/ml 25-HC for 12 or 16 h. BFA (afinal concentration of 10 µM) and a proteasome inhibitor, ALLN (a finalconcentration of 100 µM) were directly added to the medium 1 and 6 hbefore beginning the assay, respectively.

ImmunoblottingFor immunoblot analyses of SREBPs, cells were fractionated into nuclear andcytoplasmic fractions using NE-PERNuclear and Cytoplasmic Extraction Kit(Thermo Scientific). In other experiments, cells were lysed in lysis buffer(50 mM Tris-HCl [pH 7.5], 1% Triton X-100, 150 mM NaCl) containing aprotease inhibitor cocktail (EDTA-free) (Nacalai Tesque). Proteins in thefractions or the lysates were separated by using SDS-PAGE and were thenelectroblotted onto an Immobilon-P transfer membrane (Millipore). Themembrane was blocked in 5% BSA and incubated sequentially with primaryand horseradish-peroxidase–conjugated secondary antibodies. Detection wasperformed using the Chemi-Lumi One L kit (Nacalai Tesque).

Immunofluorescence analysis and visualization of lipid dropletsCells grown on coverslips were fixed with 3% paraformaldehyde for 15 min,treated with 50 mM NH4Cl for 20 min and permeabilized with 0.1% TritonX-100 for 5 min. The processed cells were blocked with 10% FBS for30 min, incubated sequentially with primary and secondary antibodies for1 h each, and then subjected to immunofluorescence analysis using anAxiovert 200 MAT microscope (Carl Zeiss) or an A1-RMP confocal laser-scanning microscope (Nikon). To visualize lipid droplets, BODIPY 493/503(10 μg/ml) was included in the secondary antibody solution.

TAG quantificationTo determine cellular TAG contents, TAGs were extracted from cells asdescribed previously by Tsuboi et al. with somemodifications (Tsuboi et al.,2004). Cells that had been pre-incubated with oleic acid as described abovewere trypsinized and collected. The collected cells were counted, and thecells were then suspended in 500 μl of isopropanol and sonicated to extractintracellular TAGs. Following centrifugation, a portion (300 μl) of thesupernatant was subjected to quantification of TAGs using a TriglycerideE-test kit (Wako Pure Chemical Industries).

Luciferase assayCells were transfected with a reporter plasmid and pGL4.74[hRluc/TK],followed by transfection with siRNAs, and then subjected to assays forfirefly and Renilla luciferase activities using the Dual-Luciferase ReporterAssay System (Promega), as described previously (Irisawa et al., 2009).

Semi-quantitative real-time PCRTotal RNA was extracted from cells using the RNeasy Mini Kit (Qiagen)and subjected to reverse transcription using a SuperScript VILO cDNASynthesis Kit (Invitrogen). The resultant cDNA was used as a template forPCR using LightCycler FastStart DNAMasterPLUS SYBR Green I (RocheApplied Science).

AcknowledgementsWe thank Toshio Kitamura and Hiroyuki Miyoshi for providing materials, KazutoshiMori for critical comments on the manuscript, and Hiroyuki Takatsu and Yohei Katohfor technical support.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsK.T., A.S., S.H. and T.F. designed and performed experiments; C.Y. and S.N.assisted with experiments; and R.S., H.-W.S. and K.N. designed experiments andprepared the manuscript.

FundingThis work was supported in part by grants from the Ministry of Education, Culture,Sports, Science and Technology of Japan [grant number 22390013 to K.N.]; theJapan Society for Promotion of Science [grant number 21113512 to K.N.]; OnoMedical Research Foundation; and Uehara Memorial Foundation.

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.164137/-/DC1

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